In cells of many organisms exposure to an environment in which oxygen is depleted relative to optimal levels induces a hypoxic response. In these hypoxic cells, activation of a transcriptional cascade involving hypoxia inducible factor (HIF) directs a series of adaptive responses that enhance oxygen delivery or limit oxygen demand. Activation of HIF in cancer and ischaemic hypoxic vascular diseases has revealed its important role in human pathology and demonstrated that manipulation of HIF activity has important therapeutic potential.
The HIF transcriptional complex comprises an αβ heterodimer, HIF-β being a constitutive nuclear protein that dimerises with oxygen regulated HIF-α subunits (Semenza, G. L. (2000) Genes Dev. 14, 19831991). The activity of HIF-α, is suppressed by oxygen-dependent modification catalysed by a series of Fe(II) and 2OG dependent dioxygenases that hydroxylate specific HIF-α residues. In the presence of oxygen in human HIF-1α, 4-hydroxylation of Pro402 or Pro564 by a set of HIF prolyl hydroxylase isozymes (PHD1-3) (Epstein et al. (2001) Cell 107, 4354; Bruick, R. K., and McKnight, S. L. (2001) Science 294, 13371340) mediates its recognition by the von Hippel-Lindau (VHL) ubiquitin ligase complex and consequent targeting for proteasomal destruction (Ivan et al, (2001) Science 292, 464468; Jaakkola et al (2001) Science 292, 468472, WO 02/074981). In a complementary mechanism FIH catalyses β-hydroxylation of HIF-1α Asn803 (Lando et al, (2002) Science 295, 858861) blocking interaction with the transcriptional co-activator p300 (Dames et al., (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 52715276; Freedman et al, (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 53675372). In hypoxia, limitation of enzymatic activity allows HIF-α to escape destruction and become transcriptionally active.
Inhibition of HIF hydroxylases strongly activates the HIF transcriptional cascade even in the presence of oxygen (Epstein et al. (2001) Cell 107, 4354). Thus, inhibition of the HIF hydroxylases results in a pro-angiogenetic response that may be used in the treatment of cardiovascular diseases/ischaemic hypoxic vascular diseases including myocardial infarction and anaemia. A problem with this approach is that the human cells contain other enzymes belonging to the same family as the HIF hydroxylases, i.e. utilising dioxygen (a cosubstrate), 2-oxoglutarate (2OG) (a cosubstrate) and Fe(II) (a cofactor). Such enzymes are exemplified by phytanoyl coenzyme A hydroxylase, procollagen prolyl-4-hydroxylase, procollagen prolyl-3-hydroxylase, gamma-butyrobetaine hydroxylase, Alk B (a DNA repair enzyme) and others including predicted 2OG oxygenases identified on the basis of sequence analyses including a sub-family related to FIH (Hewitson et al., J BIOL CHEM 277 (29): 26351-26355, 2002). It is generally agreed that it is desirable that enzyme inhibitors used as pharmaceuticals are selective for their intended target or the targets involved in producing the desired effect. A lack of selectivity can lead to toxic side effects that render particular compounds unsuitable for use in human or animal therapy. One approach to identifying compounds that are selective for the intended target is to undertake structural, mechanistic and other analyses on the intended agents and to use the information gained to aid in the preparation of selective compounds, or more selective compounds (relative to those previously known), for use as pharmaceuticals for use in humans or animals. Here we describe structural and other studies on the HIF hydroxylases that enable the design of selective inhibitors of FIH and related enzymes.